The role of lipopolysaccharide in modulating L. pneumophila biofilm formation and infection

Biofilm formation is an important survival strategy commonly employed by bacteria, which are embedded in an extracellular matrix comprising proteins, carbohydrates, lipids, and DNA. This provides protection against environmental pressures such as shear flow, host immune/inflammatory responses, and antimicrobial agents. Legionella pneumophila is a Gram-negative bacterium that inhabits natural/artificial freshwater systems within multispecies biofilms. It replicates within amoebae and ciliates but can also infect the human lung and cause Legionellosis. The Legionella collagen-like (Lcl) protein is an extracellular peripheral membrane protein, and we propose it has a fundamental role in ecology and infection of lung tissue, through mediating biofilm formation and adhesion/entry into host cells, respectively. Our recent work showed its C-terminal domain (CTD) recognises host glycosaminoglycans (GAGs) through an unusual binding mode. In our preliminary studies, we have also determined that Mg2+ binds within a negatively charged internal cavity, which results in CTD trimer rearrangement. In this state, rather than binding GAGs, the CTD interacts with the lipopolysaccharide (LPS) of L. pneumophila, and other Gram-negative bacteria commonly associated with these biofilms. In addition, our preliminary studies show how a peptidoglycan deacetylase, PgdA, is also transported to the bacterial surface where it can deacetylate L. pneumophila LPS, resulting in biofilm dissemination. Our work suggests that in aquatic environments Lcl is saturated by divalent cations, and its primary role is to promote intercellular adhesion in biofilms. When encountering GAGs in the lungs, this induces a conformational switch in the CTD, causing release of free bacteria and facilitating infection. Likewise, PgdA can also reduce bacterial aggregation by altering the hydrophobicity/acetylation of L. pneumophila LPS, and this may also inhibit binding of Lcl. The focus of this proposal is to understand how Lcl and PgdA enable L. pneumophila to switch between biofilm formation and host infection. These specific research aims will be addressed: Establish how Lcl recognises LPS Determine the mechanism of biofilm regulation by PgdA Understand how Lcl and PgdA promote multispecies biofilm growth Our preliminary data demonstrates that the Lcl/PgdA systems are highly tractable for structural and cellular investigations and these studies will provide an opportunity to unravel many of the mysteries underlying how they function. Specifically, we will: i) Identify/characterise specific LPS-derived ligands using biochemical/biophysical approaches. ii) Determine structures of Lcl CTD and PgdA/homologs with/without ligands using a combination of X-ray crystallography, NMR, and MD. iii) Analyse L. pneumophila LPS interactions in membranes using liposomes and MD. iv) Create knockout mutants in L. pneumophila 130b strain and compare phenotypes of WT, KO, and complemented strains for mono and multispecies biofilm growth. v) Provide detailed insight into the roles of Lcl and PgdA/homologs and propose models for their functional mechanism(s). vi) Test models with mutagenesis combined with biochemical and cellular assays. The proposed research programme will not only provide new insights into biofilm formation and infection in L. pneumophila but will have much wider implications for understanding the role of lipopolysaccharides during biofilm formation in other bacteria. As such these studies are expected to be intrinsically fascinating (achieving scientific advancement and new knowledge) but also have the potential to inspire new ways of combatting Legionella colonisation of water systems and infection of the human host. This proposal embraces the scientific aims of the MRC through studying disease-related proteins with a view to basic biological understanding and assisting therapeutic avenues of exploration.